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Acknowledgments Med Microbiol Immunol (Berl). 2014;203:93–9. http://dx.doi.org/ We thank Shneh Sethi for helpful advice and Sabrina Mündlein, 10.1007/s00430-013-0319-9 8. Prakash A, Sharma C, Singh A, Kumar Singh P, Kumar A, Hagen F, Grit Mrotzek, and Ahmad Saleh for excellent technical assistance. et al. Evidence of genotypic diversity among Candida auris isolates The German National Reference Center NRZMyk is funded by by multilocus sequence typing, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and amplified fragment the Robert Koch Institute from funds provided by the German length polymorphism. Clin Microbiol Infect. 2016;22:277e1–9. Ministry of Health (grant no. 1369-240). Calculations were http://dx.doi.org/10.1016/j.cmi.2015.10.022 performed on the Freiburg Galaxy server using computing 9. Magobo RE, Corcoran C, Seetharam S, Govender NP. Candida services provided by the Center of Genetic Epidemiology auris-associated candidemia, South Africa. Emerg Infect Dis. 2014;20:1250–1. http://dx.doi.org/10.3201/eid2007.131765 (Danish Technical University, Lyngby, Denmark). The Freiburg 10. Chowdhary A, Prakash A, Sharma C, Kordalewska M, Kumar A, Galaxy project is supported by the Collaborative Research Sarma S, et al. A multicentre study of antifungal susceptibility Centre 992 Medical Epigenetics (DFG grant no. SFB 992/1 patterns among 350 Candida auris isolates (2009–17) in India: 2012) and German Federal Ministry of Education and Research role of the ERG11 and FKS1 genes in azole and echinocandin resistance. J Antimicrob Chemother. 2018;73:891–9. (BMBF grant no. 031 A538A). T.D. acknowledges support http://dx.doi.org/10.1093/jac/dkx480 by the Deutsche Forschungsgemeinschaft (project no. 210879364–TRR 124/B1). Address for correspondence: Oliver Kurzai, University of Würzburg Institute for Hygiene and Microbiology, Josef-Schneider-Straße 2 / E1, About the Author Würzburg 97080, Germany; email: [email protected] Dr. Hamprecht is a clinical microbiologist at the Institute for Medical Microbiology, Immunology and Hygiene and professor for antibiotic resistance of gram-negative pathogens at the University of Cologne, Germany, and the German Centre for Infection Research (DZIF), also in Cologne. His research interests include multidrug-resistant organisms (mainly Enterobacterales and fungi), their resistance mechanisms, and the improvement of diagnostic methods. Characterization of References Clinical Isolates of 1. Satoh K, Makimura K, Hasumi Y, Nishiyama Y, Uchida K, Yamaguchi H. Candida auris sp. nov., a novel ascomycetous marneffei yeast isolated from the external ear canal of an inpatient in a and Related , Japanese hospital. Microbiol Immunol. 2009;53:41–4. http://dx.doi.org/10.1111/j.1348-0421.2008.00083.x California, USA 2. Chowdhary A, Voss A, Meis JF. Multidrug-resistant Candida auris: “new kid on the block” in hospital-associated infections? J Hosp Infect. 2016;94:209–12. http://dx.doi.org/10.1016/j.jhin.2016.08.004 Linlin Li, Katelyn Chen, Nirmala Dhungana, 3. Schelenz S, Hagen F, Rhodes JL, Abdolrasouli A, Chowdhary A, Yvonne Jang, Vishnu Chaturvedi,1 Ed Desmond2 Hall A, et al. First hospital outbreak of the globally emerging Candida auris in a European hospital. Antimicrob Resist Infect Author affiliation: California Department of Public Health, Control. 2016;5:35. http://dx.doi.org/10.1186/s13756-016-0132-5 Richmond, California, USA 4. Lockhart SR, Etienne KA, Vallabhaneni S, Farooqi J, Chowdhary A, Govender NP, et al. Simultaneous emergence of multidrug- DOI: https://doi.org/10.3201/eid2509.190380 resistant Candida auris on 3 continents confirmed by whole- genome sequencing and epidemiological analyses. Clin Infect Dis. 2017;64:134–40. http://dx.doi.org/10.1093/cid/ciw691 Talaromyces marneffei and other Talaromyces species can 5. Kohlenberg A, Struelens MJ, Monnet DL, Plachouras D; The cause opportunistic invasive fungal infections. We char- Candida auris Survey Collaborative Group. Candida auris: acterized clinical Talaromyces isolates from patients in epidemiological situation, laboratory capacity and preparedness California, USA, a non–Talaromyces-endemic area, by a in European Union and European Economic Area countries, 2013 multiphasic approach, including multigene phylogeny, ma- to 2017. Euro Surveill. 2018;23:18-00136. http://dx.doi.org/ 10.2807/1560-7917.ES.2018.23.13.18-00136 trix-assisted laser desorption/ionization time-of-flight mass 6. Jeffery-Smith A, Taori SK, Schelenz S, Jeffery K, Johnson EM, spectrometry, and phenotypic methods. We identified 10 Borman A, et al.; Candida auris Incident Management Team. potentially pathogenic Talaromyces isolates, 2 T. marneffei. Candida auris: a review of the literature. Clin Microbiol Rev. 2017;31:e00029-17. http://dx.doi.org/10.1128/CMR.00029-17 1Current affiliation: New York State Department of Health, Albany, 7. Hamprecht A, Christ S, Oestreicher T, Plum G, Kempf VA, New York, USA. Göttig S. Performance of two MALDI-TOF MS systems for the 2 identification of yeasts isolated from bloodstream infections and Current affiliation: Hawaii State Department of Health, Pearl City, cerebrospinal fluids using a time-saving direct transfer protocol. Hawaii, USA.

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alaromyces marneffei is a dimorphic fungal patho- both medium types and at both incubation temperatures. T gen that causes focal or systemic infection in immu- T. australis and T. stollii isolates also produced red pig- nocompromised persons, primarily HIV-infected patients ment by 3 days but with variations based on media or tem- (1). Many cases have been reported in travelers returning perature. At 7 days of growth, the 4 T. atroroseus isolates from areas of Southeast Asia, southern China, and eastern also showed variable red pigment production (abundant, India to which it is endemic. Other Talaromyces species weak, and absent) (Appendix). Microscopically, most iso- also have been reported to cause invasive fungal infections, lates showed biverticillate conidiophores and globose to including T. amestolkiae (2), T. purpurogenus (3,4), and T. fusiform conidia in unbranched chains. Both T. marneffei piceus (5,6). Talaromyces species are common in air, soil, isolates were from HIV-positive patients. MDL17022 was and human habitats. Clinical laboratories in areas to which from a blood sample of a 37-year-old man with a travel his- this is not endemic often do not perform identifi- tory to Southeast Asia; MDL18026 was from skin tissue of cation of T. marneffei and other Talaromyces species (2). a 36-year-old man with no available travel history. Therefore, we devised a multiphasic approach for identify- Using matrix-assisted laser desorption/ionization- ing T. marneffei and other potentially pathogenic Talaro- time-of-flight (MALDI-TOF) mass spectrometry, we myces species. generated main spectrum profiles (MSP) of Talaromyces We conducted this study during 2018. Talaromyces species following Bruker’s custom MSP and library cre- isolates from 10 human specimens were submitted to the ation standard operating procedure (https://www.bruker. Microbial Diseases Laboratory (MDL), California De- com). We extracted proteins of Talaromyces isolates us- partment of Public Health (Richmond, CA, USA), to rule ing the previously published National Institutes of Health out T. marneffei (Appendix, https://wwwnc.cdc.gov/EID/ (NIH) protocol (10). We analyzed Talaromyces spectra article/25/9/19-0380-App1.pdf). Temperature and pH are with MALDI Biotyper 4.1 software against combined da- known to influence pigment production and colony mor- tabases of the Filamentous Fungi Library 2.0 (Bruker) and phology of Talaromyces species; therefore, growth char- the NIH Mold Library (10), with and without inclusion of acteristics were observed using 2 different culture media newly created MSPs of Talaromyces species (Appendix). (Sabouraud dextrose agar, pH 5.6; and Sabouraud dextrose The threshold for species identification was >1.9; for ge- agar, Emmons, pH 6.9), incubated at 25°C and 30°C. Fun- nus identification,> 1.7. gal DNA was extracted using a previously reported method Using the combined databases of Filamentous Fungi (7). Talaromyces isolates were identified to species level Library 2.0 (Bruker) and NIH Mold Library, we identified using the internal transcribed spacer (ITS) region, partial none of the isolates to species level; results showed either no β-tubulin gene (BenA), and partial RNA polymerase II identification or -level identification. However, when largest subunit gene (RPB1) (8). The ITS and partial BenA we expanded the combined database with the MDL Mold Li- and RPB1 sequences were used to search for homologies brary, we correctly identified all Talaromyces isolates to the in GenBank and CBS databases (http://www.westerdi- species level with the best score >1.9. There were no ambig- jkinstitute.nl/collections). Multigene phylogenetic analy- uous identification results; that is, the second-best matched sis was conducted on the concatenated ITS–BenA–RPB1 species also had a high confidence score> 1.9. nucleotide sequence alignment (Appendix). A blastn search T. marneffei can be readily differentiated from other (https://blast.ncbi.nlm.nih.gov/blast) through the GenBank red pigment–producing Talaromyces species by yeast-like database, pairwise comparison alignment through the CBS colony conversion at 37°C. However, many clinical lab- database, or both showed 99%–100% homology for ITS, oratories no longer conduct yeast conversions. For those 97%–100% for BenA, and 91%–100% for RPB1 sequenc- laboratories, yellow-green colonies producing red soluble es with the best-matched sequences of known Talaromyces pigment at ≈3 days on common fungal culture media at species isolates. 25°C–30°C might indicate the need to further confirm T. Phylogenetic analysis of the Talaromyces isolates marneffei. It is difficult to distinguish Talaromyces species showed 7 genetic clades, consistent with previous descrip- only by macroscopic and microscopic examination. Mul- tions of the Talaromyces genera (9) (Figure). Species iden- tilocus sequencing, although confirmatory, might be too tification using a comparison of the ITS, BenA, and RPB1 time-consuming and expensive for routine use. Therefore, sequences with existing sequences and multigene phyloge- we identified all Talaromyces isolates to species level by netic analysis identified T. marneffei (isolates MDL17022 MALDI-TOF mass spectrometry by using an expanded da- and MDL18026), T. atroroseus (MDL17026, MDL17144, tabase with well-characterized Talaromyces strains. MDL17164, and MDL18070), T. islandicus (MDL18167), In conclusion, our results show that MALDI-TOF T. stollii (MDL18054), T. coalescens (MDL18102), and mass spectrometry is a good choice for rapid, less expen- T. australis (MDL18159). The 2 T. marneffei isolates pro- sive primary identification of Talaromyces species and duced diffuse red pigment early, by 3 days of growth, on other medically important fungal pathogens. Species-level

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Figure. Phylogenetic analysis of Talaromyces species based on concatenated nucleotide alignments of internal transcribed spacer, partial β-tubulin gene, and partial RNA polymerase II largest subunit gene regions, showing the relationship among clinical isolates from patients in California, USA (black squares), and reference Talaromyces species. The tree was constructed by the neighbor-joining method with 1,000 bootstrap replicates by using MEGA software (https://www.megasoftware. net). Bootstrap support values >70% are presented at the nodes. The tree was rooted with Trichocoma paradoxa CBS 788.83. GenBank accession numbers for newly generated sequences are MK601832–41 for the internal transcribed spacer, MK626499–508 for the β-tubulin gene, and MK626509– 518 for the RNA polymerase II largest subunit gene. CBS, Westerdijk Fungal Biodiversity Institute; MDL, Microbial Diseases Laboratory, California Department of Public Health. Scale bar indicates estimated phylogenetic divergence.

identification of Talaromyces isolates is clinically useful References for treatment of patients with underlying conditions, such 1. Ustianowski AP, Sieu TP, Day JN. marneffei infection in HIV. Curr Opin Infect Dis. 2008;21:31–6. as immunodeficiency, cancer, advanced age, and immuno- http://dx.doi.org/10.1097/QCO.0b013e3282f406ae suppressive therapy. 2. Villanueva-Lozano H, Treviño-Rangel RJ, Renpenning-Carrasco EW, González GM. Successful treatment of Talaromyces amestolkiae pulmonary infection with voriconazole in an acute lymphoblastic Acknowledgments leukemia patient. J Infect Chemother. 2017;23:400–2. We thank Zenda Berrada for her support and helpful suggestions http://dx.doi.org/10.1016/j.jiac.2016.12.017 on this study. 3. Atalay A, Koc AN, Akyol G, Cakir N, Kaynar L, Ulu-Kilic A. Pulmonary infection caused by Talaromyces purpurogenus in a The Microbial Diseases Laboratory at the California Department patient with multiple myeloma. Infez Med. 2016;24:153–7. of Public Health provided funding for this study. 4. Lyratzopoulos G, Ellis M, Nerringer R, Denning DW. Invasive infection due to Penicillium species other than P. marneffei. J Infect. 2002;45:184–95. http://dx.doi.org/10.1053/jinf.2002.1056 About the Author 5. Santos PE, Piontelli E, Shea YR, Galluzzo ML, Holland SM, Zelazko ME, et al. Penicillium piceum infection: diagnosis Dr. Li is a research scientist and a certified public health and successful treatment in chronic granulomatous disease. microbiologist at the Microbial Diseases Laboratory, California Med Mycol. 2006;44:749–53. http://dx.doi.org/10.1080/ Department of Public Health. Her primary research interests 13693780600967089 include molecular diagnosis of mycotic diseases, Valley fever, 6. Horré R, Gilges S, Breig P, Kupfer B, de Hoog GS, Hoekstra E, et al. Case report. Fungaemia due to Penicillium piceum, a member candidemia, antifungal susceptibility testing, mycobacteriology, of the Penicillium marneffei complex. Mycoses. 2001;44:502–4. metagenomics, and next-generation sequencing. http://dx.doi.org/10.1046/j.1439-0507.2001.00710.x

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7. Romanelli AM, Fu J, Herrera ML, Wickes BL. A universal DNA arathyridaria percutanea, earlier known as Rous- extraction and PCR amplification method for fungal rDNA soella percutanea in the order Pleosporales, has sequence-based identification. Mycoses. 2014;57:612–22. P http://dx.doi.org/10.1111/myc.12208 been reported to cause subcutaneous phaeohyphomycoses 8. Samson RA, Yilmaz N, Houbraken J, Spierenburg H, Seifert KA, (1,2). P. percutanea belongs to coelomycetes, a group of Peterson SW, et al. Phylogeny and nomenclature of the genus fungi in which the conidia or asexual propagules lie with- Talaromyces and taxa accommodated in Penicillium subgenus in a cavity. Parathyridaria spp. generally exist as plant Biverticillium. Stud Mycol. 2011;70:159–83. http://dx.doi.org/ 10.3114/sim.2011.70.04 saprobes; P. percutanea is the only species reported as an 9. Yilmaz N, Visagie CM, Houbraken J, Frisvad JC, Samson RA. opportunistic pathogen. Polyphasic of the genus Talaromyces. Stud Mycol. We recently observed a case of subcutaneous phaeo- 2014;78:175–341. http://dx.doi.org/10.1016/j.simyco.2014.08.001 hyphomycosis caused by P. percutanea. The patient was 10. Lau AF, Drake SK, Calhoun LB, Henderson CM, Zelazny AM. Development of a clinically comprehensive database and a simple a 33-year-old man who had ACTH-dependent Cushing’s procedure for identification of molds from solid media by disease with 2 cutaneous lesions, one under the left axilla matrix-assisted laser desorption ionization-time of flight mass and the other on the ulnar aspect of the left forearm, that spectrometry. J Clin Microbiol. 2013;51:828–34. http://dx.doi.org/ had progressed slowly over 3 years (Appendix Figure 1, 10.1128/JCM.02852-12 panel A, https://wwwnc.cdc.gov/EID/article/25/9/19-0383- Address for correspondence: Ed Desmond, State Laboratories Division, App1.pdf). Direct microscopy of a biopsy sample taken Hawaii State Department of Health, 2725 Waimano Home Rd, Pearl from the left forearm lesion revealed dematiaceous septate City, HI 96782, USA; email: [email protected] hyphae with irregular hyphal swellings (Appendix Fig- ure 1, panel B). Colonies on Sabouraud’s dextrose agar at 25°C were flat, spreading with sparse aerial hyphae after 1 week, and later turned to cottony greenish-black growth (Appendix Figure 1, panel C). Lactophenol cotton blue mount revealed nonsporulating dematiaceous hyphae with chlamydospores (Appendix Figure 1, panel D). Several at- tempts to induce sporulation (on oatmeal agar and malt ex- tract agar) failed. Histopathologic examination (Appendix Figure 1, panels E–G) showed neutrophilic infiltration with Parathyridaria percutanea fungal hyphae, nodular swellings on Giemsa stain, and and Subcutaneous black hyphae on Grocott-Gomori’s methamine silver stain. Phaeohyphomycosis We identified the fungus as Roussoella percutanea of the order Pleosporales, later renamed P. percutanea, by PCR sequencing of the internal transcribed spacer (ITS) and 28S Shivaprakash M. Rudramurthy,1 Megha Sharma,1 regions of ribosomal DNA, as described previously (3). ITS Nandini Sethuraman, Pinaki Dutta, sequencing of our strain NCCPF104001 (GenBank acces- Bansidhar Tarai, Jayanthi Savio, Amanjit Bal, sion nos. MG708109 [by ITS] and MG708116 [by 28S]) had Usha Kalawat, Arunaloke Chakrabarti 99.8% identity with CBS128203 (type strain, GenBank ac- cession no. KF322117) and CBS868.95 (GenBank accession Author affiliations: Postgraduate Institute of Medical Education no. KF322118), whereas 28S sequences had 100% identity and Research, Chandigarh, India (S.M. Rudramurthy, M. Sharma, with CBS128203 (GenBank accession no. KF366448) and N. Sethuraman, P. Dutta, A. Bal, A. Chakrabarti); Apollo Hospitals, CBS868.95 (GenBank accession no. KF366449) (Appendix Chennai, India (N. Sethuraman); Max Super Speciality Hospitals, Figure 2, panels A. B). The patient refused further treatment New Delhi, India (B. Tarai); St. Johns Medical College and in the hospital and left against medical advice. Research Institute, Bengaluru, India (J. Savio); Sri Venkateshwara We screened all the isolates deposited in our National Institute of Medical Sciences, Tirupati, India (U. Kalawat) Culture Collection of Pathogenic Fungi (NCCPF, Chandi- DOI: https://doi.org/10.3201/eid2509.190383 garh) and characterized them phenotypically as Pleospora- les. Of 7 such isolates, we identified 4 asP. percutanea by se- Parathyridaria percutanea is an emerging fungus causing quencing (Table, https://wwwnc.cdc.gov/EID/article/25/9/ subcutaneous phaeohyphomycoses in renal transplant re- 19-0383-T1.htm). We further subjected these isolates to cipients in India. We identified P. percutanea from a patient phylogenetic analysis of ITS and large ribosomal subunit with subcutaneous phaeohyphomycosis. From our culture (28S) of the rDNA using MEGA software version 6 (https:// collection, we identified the same fungus from 4 similar pa- megasoftware.net) (3). The strains identified as P. percuta- tients. We found 5 cases previously described in literature. nea clustered together with the ITS and 28S sequences of 1These first authors contributed equally to this article. CBS12608 and CBS868.95 strains, the other 2 P. percutanea

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